Bottom Line:
A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not.A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity.The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.

ABSTRACTBacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H(2)O(2) and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.

pone-0038054-g007: Effect of ceramide on activation of mouse macrophages.A) Mouse macrophages were incubated with various concentrations of Bc-SMase at 37°C for 60 min. Ceramide was phosphorylated by 1, 2-diacylglycerol kinase and separated by reverse-phase TLC. Values represent the mean ± SEM; n = 3; * P<0.01 compared with Bc-SMase-untreated cells. (B) Macrophages pretreated with BODIPY-SM were incubated without (left) or with (right) Bc-SMase at 37°C for 60 min. (C) Mouse macrophages pretreated with BODIPY-SM were incubated with or without Bc-SMase at 37°C for 60 min. The cells were treated with paraformaldehyde, and stained with Cy3-coupled anti-ceramide anti-bodies. (D) Representative recovery curves for the diffusion of BODIPY fluorescence following Bc-SMase treatment (broken line, ceramide-rich domain) or without treatment (solid line, ceramide-poor domain) are shown. A representative result from one of ten experiments is shown.

Mentions:
To determine the amount of ceramide formed in the macrophages treated with Bc-SMase, macrophages were incubated with Bc-SMase at 37°C for 30 min. The lipids extracted from the treated cells were phosphorylated by diacylglycerol kinase from Escherichia coli, and developed by reverse-phase thin layer chromatography (TLC). The level of ceramide in the cells treated with Bc-SMase increased in a dose-dependent manner (Fig. 7A). Using confocal microscopy, Montes et al. found that phospholipase C from P. aeruginosa caused the formation of ceramide-rich domains in biological membranes [28]. We reported that Bc-SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation [19]. We investigated whether treatment of macrophages with Bc-SMase results in the local accumulation of BODIPY-ceramide formed in membranes of cells preincubated with BODIPY FL-C12-SM (BODIPY-SM). Fig. 7B (left) shows that the fluorescent substance in membranes of macrophages preincubated with BODIPY-SM was not localized. However, when BODIPY-SM -incubated macrophages were treated with Bc-SMase, the local accumulation of the fluorescent substance was found on membranes of the cells, as shown by the white arrows (Fig. 7B, right). To test whether the site where the substance accumulates coincides with a ceramide-rich site, BODIPY–SM-preincubated membranes treated with Bc-SMase were analyzed using Cy3-labeled anti-ceramide antibody. As shown in Fig. 7C, the distribution of the fluorescence of the antibody was different from that of BODIPY–SM in the untreated cells. In the case of BODIPY–SM-preincubated macrophages treated with Bc-SMase, the location of the fluorescence of Cy3-anti-ceramide antibody was consistent with that of BODIPY, as shown in Fig. 7B, suggesting that BODIPY-ceramide formed from BODIPY-SM in the macrophages treated with Bc-SMase is mostly located in ceramide-rich domains. Klein et al. reported that membrane fluidity of cells was evaluated by measurement of lateral diffusion of fluorescence-labeled SM by FRAP with a confocal laser microscopy [29]. A FRAP analysis revealed that the recovery of effective diffusion for the fluorescence of BODIPY in ceramide-rich domains of the macrophages treated with Bc-SMase decreased to about 70–80%, compared with that of BODIPY-SM in the untreated cells (Fig. 7D). It therefore appears that the Bc-SMase-induced formation of ceramide from SM in membranes of macrophages results in a decrease in the fluidity of membranes.

pone-0038054-g007: Effect of ceramide on activation of mouse macrophages.A) Mouse macrophages were incubated with various concentrations of Bc-SMase at 37°C for 60 min. Ceramide was phosphorylated by 1, 2-diacylglycerol kinase and separated by reverse-phase TLC. Values represent the mean ± SEM; n = 3; * P<0.01 compared with Bc-SMase-untreated cells. (B) Macrophages pretreated with BODIPY-SM were incubated without (left) or with (right) Bc-SMase at 37°C for 60 min. (C) Mouse macrophages pretreated with BODIPY-SM were incubated with or without Bc-SMase at 37°C for 60 min. The cells were treated with paraformaldehyde, and stained with Cy3-coupled anti-ceramide anti-bodies. (D) Representative recovery curves for the diffusion of BODIPY fluorescence following Bc-SMase treatment (broken line, ceramide-rich domain) or without treatment (solid line, ceramide-poor domain) are shown. A representative result from one of ten experiments is shown.

Mentions:
To determine the amount of ceramide formed in the macrophages treated with Bc-SMase, macrophages were incubated with Bc-SMase at 37°C for 30 min. The lipids extracted from the treated cells were phosphorylated by diacylglycerol kinase from Escherichia coli, and developed by reverse-phase thin layer chromatography (TLC). The level of ceramide in the cells treated with Bc-SMase increased in a dose-dependent manner (Fig. 7A). Using confocal microscopy, Montes et al. found that phospholipase C from P. aeruginosa caused the formation of ceramide-rich domains in biological membranes [28]. We reported that Bc-SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation [19]. We investigated whether treatment of macrophages with Bc-SMase results in the local accumulation of BODIPY-ceramide formed in membranes of cells preincubated with BODIPY FL-C12-SM (BODIPY-SM). Fig. 7B (left) shows that the fluorescent substance in membranes of macrophages preincubated with BODIPY-SM was not localized. However, when BODIPY-SM -incubated macrophages were treated with Bc-SMase, the local accumulation of the fluorescent substance was found on membranes of the cells, as shown by the white arrows (Fig. 7B, right). To test whether the site where the substance accumulates coincides with a ceramide-rich site, BODIPY–SM-preincubated membranes treated with Bc-SMase were analyzed using Cy3-labeled anti-ceramide antibody. As shown in Fig. 7C, the distribution of the fluorescence of the antibody was different from that of BODIPY–SM in the untreated cells. In the case of BODIPY–SM-preincubated macrophages treated with Bc-SMase, the location of the fluorescence of Cy3-anti-ceramide antibody was consistent with that of BODIPY, as shown in Fig. 7B, suggesting that BODIPY-ceramide formed from BODIPY-SM in the macrophages treated with Bc-SMase is mostly located in ceramide-rich domains. Klein et al. reported that membrane fluidity of cells was evaluated by measurement of lateral diffusion of fluorescence-labeled SM by FRAP with a confocal laser microscopy [29]. A FRAP analysis revealed that the recovery of effective diffusion for the fluorescence of BODIPY in ceramide-rich domains of the macrophages treated with Bc-SMase decreased to about 70–80%, compared with that of BODIPY-SM in the untreated cells (Fig. 7D). It therefore appears that the Bc-SMase-induced formation of ceramide from SM in membranes of macrophages results in a decrease in the fluidity of membranes.

Bottom Line:
A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not.A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity.The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.

ABSTRACTBacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H(2)O(2) and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.